Welcome to the ICOPA 2022 Online Program

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Blastocystis is frequently reported in fecal samples from animals and humans worldwide, and a variety of subtypes (STs) have been observed in wild and domestic animals. In Colombia, during the last decade, our group has focused in unveiling the molecular epidemiology of Blastocystis using conventional markers as direct sanger sequencing of the 18s. In this study, we characterized the frequency and subtypes of Blastocystis in fecal samples of humans, domestic and wild animals using conventional sanger sequencing and the use of Next Generation Sequencing.

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Blastocystis is the most commonly occurring microbial eukaryote in the gastrointestinal tract of humans and other animals. The protist is currently classified into 28 subtypes (STs) based on the small subunit ribosomal RNA gene reflecting its genetic heterogeneity. Though Blastocystis has been studied for almost a century, our understanding regarding its prevalence/occurrence in various hosts and the environment and consequently its transmission dynamics remains inadequate. Over the past few years, we have been investigating Blastocystis in rural communities in lower-middle income (LMIC) countries under the One Health umbrella. Our projects have focused on surveying human, other animal and environmental samples using a combination of both morphological and molecular approaches. We evaluate Blastocystis genetic diversity and its distribution, occurrence and transmission dynamics at the household and community level. Our findings so far are consistent with transmission among humans and between domesticated animals and humans being not as frequent as expected. This raises questions on the role of wildlife and the environment on transmission ofBlastocystis and we recommend for the two to be included in future studies. Using this type of holistic analysis advances our understanding on various aspects of this poorly understood, yet frequent gut resident.

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Glycolysis is a well-conserved cytosolic pathway that converts glucose into pyruvate, which is transported via pyruvate carrier into mitochondria and enters the TCA cycle. It was shown that in Blastocystis the second part of glycolysis is localised in mitochondria and the pyruvate carrier is missing. Therefore, for Blastocystis to perform glycolysis, there has to be another transporter for glycolytic intermediates to trespass the mitochondrial inner membrane. We aim to identify which glycolytic intermediate is transported and also to characterise the novel glycolytic transporter.

Using bioinformatics, we identified a group of Stramenopile-specific transporters which are not present in other eukaryotes. The purified proteins were used for thermostability assays to screen for potential substrates. The substrates which increased the thermostability were used in transport assays. We show that while the addition of glycolytic intermediates has no effect on the thermostability of our control proteins, it has a stabilising effect on Blastocystis-specific transporters. We also show their ability to transport glycolytic intermediates by measuring the uptake of radiolabelled substrates into proteoliposomes. It indicates that Stramenopiles adapted their transporters in order to transport glycolytic intermediates.

We show that Blastocystis transporters can bind glycolytic intermediates and are able to transport them across the membrane. Given that these transporters are Stramenopile‑specific, they could be exploited as drug targets not only for Blastocystis but also for economically important pathogens such as Saprolegnia or Phytophthora.

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Blastocystis is one of the most common intestinal parasites in humans in developing and developed countries in the world. However, the role of Blastocystis as a pathogen is still controversial, mainly because it is found in both patients suffering from intestinal symptomatology, such as diarrhea, flatulence, bloating, or abdominal discomfort, as well as in healthy people. Molecular characterization to identify subtypes present in samples is critical to unravel Blastocystis epidemiology and to characterize subtype level differences in host specificity, transmission, public health significance, and pathogenicity. Mixed subtype infections are often overlooked in molecular studies of Blastocystis, and a better characterization of these infections is needed to fully understand the epidemiology of Blastocystis. A high degree of genetic diversity has been found among Blastocystis isolates based on nucleotide differences in the small subunit (SSU) of the ribosomal RNA (rRNA) gene. Accurate assessment of Blastocystis subtype (ST) diversity is crucial to understanding epidemiology and sources of Blastocystis transmission to humans. The next-generation sequencing (NGS) improves the detection of mixed subtype infections and low abundance STs. NGS technology contributes to our understanding of the epidemiology of Blastocystis infection in humans and its zoonotic potential. In addition, it can be used to shape future studies which aim to better characterize the transmission pathways. Moreover further case-control studies implementing high-resolution molecular tools are necessary to understand the role of Blastocystis in health and disease. NGS data will be provided great benefit to the One Health approach for this protist.

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We determined the occurrence and genetic diversity of Blastocystis sp. in wild ungulates in the Iberian Peninsula including nationwide sample collections from Portugal (n = 222) and Spain (n = 1,058). Faecal samples from three wild ungulate species in Portugal (genera Capreolus, n = 38; Cervus, n = 79; and Sus, n = 105) and eight wild ungulate species in Spain (genera Ammotragus, n = 20; Capra, n = 89; Capreolus, n = 93; Cervus, n = 329; Dama, n = 96; Ovis, n = 10; Rupicapra, n = 62; and Sus, n = 359) were retrospectively investigated by PCR. Next generation amplicon sequencing (NGS) data was used to assess presence of Blastocystis and subtype diversity within a sample. Blastocystis sp. was identified in 37.4% (95% CI: 31.0–43.8%) of the Portuguese samples, with minimum and maximum rates detected in roe deer (31.6%) and red deer (40.5%), respectively. In Spain, 8.7% (95% CI: 7.1–10.4%) of the samples were Blastocystis-positive. Minimum and maximum rates were identified in Iberian wild goats (3.4%) and roe deer (12.9%), respectively. Thirteen Blastocystis subtypes (ST2, ST5, ST10, ST13, ST14, ST15, ST21, ST23, ST24, ST25, ST26, ST30, and ST31) were identified by NGS circulating in the wild ungulate populations in the Iberian Peninsula. The presence of zoonotic Blastocystis ST2 and ST5 in wild ungulates suggests that they could be reservoirs for human infection with this parasite. Blastocystis mixed subtype infections were frequent (42.3%, 74/175), with some animals harbouring up to 11 subtypes. Marked differences in Blastocystis subtype distribution according to host species were observed between countries. NGS was a powerful tool to discern underrepresented or mixed subtype infections that were undetectable or unreadable by Sanger sequencing.